Seeinstructions): Alloantibodies are induced antibodies that limit the availability and success of heart and lung allotransplantation. In the presently funded period, we have demonstrated that mice presensitized to alloantigens can be rendered tolerant to these antigens using a non-myeloablative regimen for mixed chimerism induction. However, chimerism induction is markedly impeded by the presence of anti-donor alloantibodies. In the proposed renewal of this project, we will address this problem in both the mouse model and a novel humanized mouse model that we have developed, with the goal of developing a clinically relevant mixed chimerism regimen that will overcome both T and B cell sensitization to the donor. We will: Aim 1: Identify the cell types producing anti-donor alloantibodies in presensitized mice and develop methods of depleting them. This information will then be applied to the development of a non-myeloablative mixed chimerism regimen that overcomes both pre-sensitized T cell and alloantibody responses, allowing vascularized heart transplantation in presensitized hosts; Aim 2: Identify the human cell types with potential to produce alloantibodies in presensitized humanized mice and develop methods of depleting them. We will develop a protocol for mixed allogeneic chimerism induction in humanized mice and extend this approach to the allopresensitized setting. Natural antibodies (Nab) limit the success of both allogeneic and xenogeneic transplantation. Nab recognition of blood group antigens and of autoantigens exposed by ischemia-reperfusion injury can induce hyperacute and acute humoral rejection and promote other forms of rejection. We have demonstrated grant that mixed chimerism leads to tolerance of anti-carbohydrate (aGal) Nab-producing B cells by an anergy mechanism that is later followed by deletion of donor-reactive B cells. This tolerance is dependent on complement receptor expression by non-hematopoietic cells of the recipient. We hypothesize that tolerance of the B-1b B cells producing Nabs requires interactions with splenic follicular dendritic cells (FDCs) that pick up immune complexes via their complement receptors. Aim 3: Determine the role of secreted Ig and complement receptors in the tolerization of natural antibody-producing cells. We will address the hypothesis that immune complexes formed by natural antibodies to donor antigens and complement components target the Nab-producing B cells to FDCs in the spleen, providing a critical activation step that renders the cells susceptible to subsequent anergy and ultimate deletion upon repeated encounter with cell surface-bound antigen on donor-derived hematopoietic cells. _^_____ RELEVANCE (Seeinstructions): Together, the studies in this project will allow the improved application of the mixed chimerism approach to overcoming antibody-dependent as well as cellular rejection to the large animal models in this Program Project grant. Through the pipeline of small to large animal models, these studies will ultimately lead to the succesful induction of donor-specific T and B cell tolerance in human recipients with pre-existing antibodies to the heart or lung donor.